Vaccines have had profound and long lasting effects on world health. Small pox has been eradicated, polio is near elimination, and diseases such as diphtheria, measles, mumps, pertussis, and tetanus are contained. Nonetheless, microbes remain major killers with current vaccines addressing only a handful of the infections of man and his domesticated animals. Common infectious diseases for which there are no vaccines cost the United States $120 billion dollars per year (Robinson et al., 1997). In first world countries, emerging infections such as immunodeficiency viruses, as well as reemerging diseases like drug resistant forms of tuberculosis, pose new threats and challenges for vaccine development. The need for both new and improved vaccines is even more pronounced in third world countries where effective vaccines are often unavailable or cost-prohibitive. Recently, direct injections of antigen-expressing DNAs have been shown to initiate protective immune responses.
DNA-based vaccines use bacterial plasmids to express protein immunogens in vaccinated hosts. Recombinant DNA technology is used to clone cDNAs encoding immunogens of interest into eukaryotic expression plasmids. Vaccine plasmids are then amplified in bacteria, purified, and directly inoculated into the hosts being vaccinated. DNA typically is inoculated by a needle injection of DNA in saline, or by a gene gun device that delivers DNA-coated gold beads into skin. The plasmid DNA is taken up by host cells, the vaccine protein is expressed, processed and presented in the context of self-major histocompatibility (MHC) class I and class II molecules, and an immune response against the DNA-encoded immunogen is generated.
The historical foundations for DNA vaccines (also known as “genetic immunization”) emerged concurrently from studies on gene therapy and studies using retroviral vectors. Gene therapy studies on DNA delivery into muscle revealed that pure DNA was as effective as liposome-encapsulated DNA at mediating transfection of skeletal muscle cells (Wolff et al., 1990). This unencapsulated DNA was termed “naked DNA,” a fanciful term that has become popular for the description of the pure DNA used for nucleic acid vaccinations. Gene guns, which had been developed to deliver DNA into plant cells, were also used in gene therapy studies to deliver DNA into skin. In a series of experiments testing the ability of plasmid-expressed human growth hormone to alter the growth of mice, it was realized that the plasmid inoculations, which had failed to alter growth, had elicited antibody (Tang, De Vit, and Johnston, 1992). This was the first demonstration of the raising of an immune response by an inoculated plasmid DNA. At the same time, experiments using retroviral vectors, demonstrated that protective immune responses could be raised by very few infected cells (on the order of 104-105). Direct tests of the plasmid DNA that had been used to produce infectious forms of the retroviral vector for vaccination, performed in an influenza model in chickens, resulted in protective immunizations (Robinson, Hunt, and Webster, 1993).
HIV-1 is projected to infect 1% of the world's population by the year 2000, making vaccine development for this recently emergent agent a high priority for world health. Preclinical trials on DNA vaccines have demonstrated that DNA alone can protect against highly attenuated HIV-1 challenges in chimpanzees (Boyer et al., 1997), but not against more virulent SIV challenges in macaques (Lu et al., 1997). A combination of DNA priming plus an envelope glycoprotein boost has raised a neutralizing antibody-associated protection against a homologous challenge with a non-pathogenic chimera between SIV and HIV (SHIV-IIIb) (Letvin et al., 1997). More recently, a comparative trial testing eight different protocols for the ability to protect against a series of challenges with SHIV-s (chimeras between simian and human immunodeficiency viruses) revealed the best containment of challenge infections by an immunization protocol that included priming by intradermal inoculation of DNA and boosting with recombinant fowl pox virus vectors (Robinson et al., 1999). This containment of challenge infections was independent of the presence of neutralizing antibody to the challenge virus. Protocols which proved less effective at containing challenge infections included immunization by both priming and boosting by intradermal or gene gun DNA inoculations, immunization by priming with intradermal or gene gun DNA inoculations and then boosting with a protein subunit; immunization by priming with gene gun DNA inoculations and boosting with recombinant fowl pox virus, immunization with protein only, and immunization with recombinant fowl pox virus only (Robinson et al, 1999). Early clinical trials of DNA vaccines in humans have revealed no adverse effects (MacGregor et al., 1996) and the raising of cytolytic T-cells (Calarota et al., 1998). A number of studies have screened for the ability of co-transfected lymphokines and co-stimulatory molecules to increase the efficiency of immunization (Robinson and Pertmer, in press).
Disadvantages of DNA vaccine approaches include the limitation of immunizations to products encoded by DNA (e.g., proteins) and the potential for atypical processing of bacterial and parasitic proteins by eukaryotic cells. Another significant problem with existing approaches to DNA vaccines is the instability of some vaccine insert sequences during the growth and amplification of DNA vaccine plasmids in bacteria. One possible cause of instability is exposure during plasmid growth of secondary structures in vaccine inserts or the plasmid backbone that can be recognized by bacterial endonucleases.
A need exists, therefore, for DNA expression vectors that exhibit improved stability in bacterial hosts and may be safely used in animals, including humans; for eukaryotic expression of immunogenic proteins useful as vaccines against a variety of infectious diseases, including HIV-1.